Bilirubin is a reddish yellow substance found in biological substances such as blood serum, and has the empirical formula: C.sub.33 H.sub.36 N.sub.4 O.sub.6
Its presence in serum at too high a level indicates jaundice and its measurement is used as a liver function test.
One standard assay used for the quantitive determination of bilirubin is the Jendrassik-Grof method which uses as the assay composition an aqueous solution of caffeine, a benzoate salt, an acetate salt and diazonium salt of sulfanilic acid formed by reacting sulfanilic acid and sodium nitrite in diluted hydrochloric acid. Bilirubin present in the serum reacts with the diazotized sulfanilic acid to form a chromophore in which the caffeine and benzoate and acetate salts cooperate to serve as an activator and speed the reaction.
The problem with the solution used is its stability. It is initially supplied as a three component system. One contains caffeine, the benzoate salt and the acetate salt. The second contains sulfanilic acid and hydrochloric acid. The third reagent contains sodium nitrite and is the least stable of the three. When the three components are combined, stability is less than 8 hours.
It would be desirable, therefore, to have a stable single reagent system which enables the measurement of bilirubin. Such a system would be lower in cost as three different components need not be packaged. It would also reduce the chance of human error which can occur in combining three components to make an assay reagent. This is because better quality control exist when all components can be added to form a single reagent in quite precise amounts. Such a reagent of adequate shelf life has not heretofore been proposed in the art in ready to use liquid form.